Nuclear localization and the 25 kDa C-terminus is essential for subsequent mitochondrial import of NOA1.

<p>(A) Immunofluorescence staining of different Flag tagged NOA1 proteins transfected into C2C12 cells reveals requirement of the NLS and the C-terminus of NOA1 for mitochondrial import. Deletion of the MTS abolishes mitochondrial localization leading to accumulation of NOA1 in the nucleus. Mutation of the NLS in the ΔMTS mutant confirms that the NLS is necessary for nuclear localization. Mitochondria were identified by co-staining for Tom20. (B) Quantitative evaluation of the subcellular dynamics of wild type and mutant NOA1 protein. Wild type NOA1 protein shows a predominant “Mitochondrial” localization. ΔMTS-NOA1 shows a predominant “nucleus” accumulation. NOA1-NLS mutants show a mixed localization as indicated by the term “aggregated”. Wild type NOA1 is dynamically distributed between nucleus and mitochondria. Leptomycin-B treatment prevents nuclear export and subsequent mitochondrial import of wild type NOA1. Mutation of the NLS prevents nuclear accumulation of NOA1 after leptomycin-B treatment. (C) N-terminal tagging prevents import of NOA1 into mitochondria of NIH 3T3 cells by masking the MTS. (D) A Western blot analysis of N-terminally (HA tag) and C-terminally (Flag tag) modified versions of NOA2 is shown. (E) Deletion of the RNA-binding domain containing C-terminus prevents mitochondrial import in NIH 3T3 cells. Western blot analysis demonstrated strongly reduced processing of the MTS after truncation of the C-terminus. Scale bars: 10 µm.